| Hepatocellular carcinoma(HCC)is one of the most common malignant tumors,the number of HCC incidence and death in C hina that ranks first in the world.At present,the treatment methods of HCC include surgery,interventional therapy,chemotherapy,radiotherapy.However,most of the patients have been diagnosed in the late stage because of early HCC is not obvious symptoms.Advanced HCC is not suitable for surgical treatment,so chemotherapy is the main treatment in this time.C urrently,HCC has appeared serious resistance to chemotherapy drugs,causing chemotherapeutic effect was not ideal.We must prevent and reverse the drug resistance of HCC when chemotherapy,but there is still no effective way to prevent and reverse the drug resistance.Therefore,clarifying the mechanism of drug resistance in order to find effective intervention targets to provide effective intervention methods,which is the key to improve the efficacy of HCC chemotherapy.The extracellular acidic microenvironment is one of the main characteristics of solid tumors,which is caused by hypoxia,high levels of glycolysis and insufficient blood perfusion.It has been found that the extracellular acid microenvironment can mediate tumor resistance.However,how does this acidic signal be deliver ed into the cell and how does it mediate tumor resistance? ASIC1 a is a subunit of acid sensing ion channels(ASICs),which acts as an acid sensor on the cell membrane and is activated in the extracellular acidic environment,and then it delivers the signal of low p H into the cell.It has been found that ASIC1 a is closely involved in the development of tumor.Whether does the extracellular acidic microenvironment activate ASIC1 a,and then transfer the acidic signal into the cell to further mediate the resistance of HCC? Therefore,we explore whether the extracellular acidic microenvironment activates ASIC1 to mediate hepatocellular carcinoma resistance and its mechanism.Objectives: To investigate the expression of ASIC1 a in resistant HCC cell lines Bel7402/FU,Hep G2/ADM and human HCC tissues.To further investigate whether ASIC1 a mediates the drug resistance of HCC in the acidic environment,and whether ASIC1 a mediates HCC resistance by regulating the Ca2 + /PI3K/AKT signaling pathwayMethods : 1.The expression of ASIC1 a in 8 pairs of human normal liver tissues and HCC tissues was detected by Western blotting.2.The expression of ASIC1 a was detected in human resistant HCC cells Bel7402/FU and Hep G2/ADM by Western blotting.3.MTT assay was used to detect the drug resistance of human resistant HCC cells Bel7402/FU to 5-FU and the drug resistance of Hep G2/ADM to ADM.4.The chemosensitivity of Bel7402/FU cells to 5-FU and chemosensitivity of H-ep G2/ADM cells to ADM was detected by MTT assay at 3,6,12,24 and 48 hours after the inhibition of ASIC1 a by 25,100,400 ?? amiloride.5.Using 25,100,400 ?? amiloride to inhibit ASIC1 a,the chemosensitivity of Bel7402/FU cells to 5-FU and chemosensitivity of Hep G2/ADM cells to ADM was detected by colony formation assay after 24 and 48 hours.6.Using ASIC1 a sh RNA lentivirus to infect human resistant HCC Bel7402/FU a-nd Hep G2/ADM.The expression of ASIC1 a was detected in Bel7402/FU an-d Hep G2/ADM cells by Western blotting after lentivirus infection,so as to screen the successful silencing cel s.7.The chemosensitivity of Bel7402/FU-sh RN A cells to 5-FU and the chemosens-itivity of Hep G2/ADM-sh RNA cel s to ADM were detected by MTT assay.8.The Bel7402/FU-sh RNA and Hep G2/ADM-sh RNA cells was treated with 25,100,400 ?? amiloride for 24 and 48 hours.The chemosensitivity of Bel740-2/FU-sh RNA cells to 5-FU and the chemosensitivity of Hep G2/ADM-sh RNA cel s to ADM were detected by MTT assay.9.Using ASIC1 a gene lentivirus to infect human HCC cells Bel7402 and Hep G-2.The expression of ASIC1 a was detected in Bel7402 and Hep G2 cells by Western blotting after lentivirus infection,so as to screen the successful overe-xpression of cel s.10.The chemosensitivity of Bel7402-ASIC1 a cells to 5-FU and the chemosensitiv-ity of Hep G2-ASIC1 a cel s to ADM were detected by MTT assay.11.The Bel7402-ASIC1 a and Hep G2-ASIC1 a cells were treated with 25,100,400 ?? amiloride for 24 and 48 hours.The chemosensitivity of Bel7402-ASIC1 a cells to 5-FU and the chemosensitivity of Bel7402-ASIC1 a cells to ADM w-ere detected by MTT assay.12.The calcium was label by Fluo-3 AM in Bel7402/FU and Hep G2/ADM cells,then the intracellular calcium levels were measured by flow cytometry.13.The calcium was label by Fluo-3 AM in Bel7402/FU and Hep G2/ADM cells,then laser confocal microscopy was used to detect whether ASIC1 a mediates calcium influx.14.The expression of PI3 K and p-AKT in Bel7402/FU-sh RNA and Hep G2/ADMsh RNA cel s was detected by Western blotting.15.The expression of PI3 K and p-AKT were detected in Bel7402/FU and Hep G2/ADM cell by Western blotting.After wortmannin and MK-2206 2HC l were used to inhibit PI3K/AKT pathway,the chemosensitivity of Bel7402/FU cells to 5-FU and chemosensitivity of Hep G2/ADM cells to ADM was detected by MTT assay.Results: 1.Compared with normal liver tissue,the expression of ASIC1 a was significantl-y increased in human HCC tissues.2.Compared with normal human liver cell L-02,human hepatoma cell Bel7402 or Hep G2,the expression of ASIC1 a was significantly increased in resistant HCC cell lines Bel7402/FU or Hep G2/ADM.3.Inhibition of ASIC1 a activity significantly enhanced the C hemosensitivity of Bel7402/FU to 5-FU and the Chemosensitivity of Hep G2/ADM to ADM.4.Silencing of the ASIC1 a gene can significantly enhance the C hemosensitivity of Bel7402/FU to 5-FU and the Chemosensitivity of Hep G2/ADM to ADM.5.Overexpression of the ASIC1 a gene can significantly enhance the C hemoresist-ance of Bel7402 to 5-FU and the Chemoresistance of Hep G2 to ADM.6.Compared with normal normal human liver cell L-02,human hepatocellular c-arcinoma cells Bel7402 or Hep G2,calcium level was significantly increased in human hepatocellular carcinoma cel s Bel7402/FU or Hep G2/ADM.7.ASIC1 a mediate the influx of calcium in resistant HCC cells Bel7402/FU and Hep G2/ADM.8.Silencing of ASIC1 a gene inhibit PI3K/AKT signaling pathway.9.Inhibition of PI3K/AKT signaling pathway ca n significantly enhance the C hemosensitivity of Bel7402/FU to 5-FU and the C hemosensitivity of Hep G2/AD-M to ADM.Conculsions:1.ASIC1 a mediates the drug resistance of human hepatocellular carcinoma.2.ASIC1 a inhibits the drug resistance of human hepatocellular carcinoma by reg-ulating Ca2 +/PI3K/AKT signaling pathway. |
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